DESCRIPTION: DNA topoisomerases are the targets for a wide range of clinically useful anti-cancer drugs. Although the basic biochemical reaction pathway for the topoisomerases is fairly well understood, there is still little information available regarding the mechanism of action of the anti-topoisomerase drugs in generating the stable covalent complex that is necessary for cell killing. Studies are proposed to carry out a detailed mutational analysis of eukaryotic topoisomerase II, with the goal of identifying sites in the enzyme which interact with complex stabilizing topoisomerase II inhibitors. Yeast is used as a model system for analyzing the effects of mutant proteins, and for overexpression of the mutant proteins. The research strategy involves the construction of novel topoisomerase mutants, including mutants that are hypersensitive to anti-topoisomerase drugs. In addition to etoposide, studies will focus on other agents with different types of interaction with topoisomerase II, such as ICRF-187, which is a catalytic inhibitor of the enzyme. Mutations will also be introduced into the human topoisomerase II alpha and be at genes to verify that the effects of mutations are similar among the yeast enzymes and the human proteins. A new system is being developed to investigate whether mutations in topoisomerase play a role in the development of acquired drug resistance. Recent findings indicating that topoisomerase I may be an important determinant in cell killing by other DNA damaging agents have led to studies of how cells may modulate topoisomerases following DNA damage. This work is designed to provide insight into the biochemical mechanisms of anti-topoisomerase drug action, and may be useful in the development of new and more effective topoisomerase inhibitors.